Mode of carbon gain and fungal associations of Neuwiedia malipoensis within the evolutionary early diverging orchid subfamily Apostasioideae.

BACKGROUND AND AIMS: The earliest diverging orchid lineage Apostasioideae consists only of two genera: Apostasia and Neuwiedia. Previous report of Apostasia nipponica indicated a symbiotic association with an ectomycorrhiza-forming Ceratobasidiaceae clade and partial utilization of fungal carbon during the adult stage. However, the trophic strategy of Neuwiedia throughout its development remains unidentified. To further improve our understanding of mycoheterotrophy in the Apostasioideae, this study focused on Neuwiedia malipoensis examining both the mycorrhizal association and the physiological ecology of this orchid species across various development stages. METHODS: We identified the major mycorrhizal fungi of N. malipoensis protocorm, leafy seedling and adult stages using molecular barcoding. To reveal nutritional resources utilized by N. malipoensis, we compared stable isotope natural abundance (δ13C, δ15N, δ2H, δ18O) of different developmental stages to autotrophic reference plants. KEY RESULTS: Protocorms exhibited an association with saprotrophic Ceratobasidiaceae rather than ectomycorrhiza-forming Ceratobasidiaceae and 13C signature was characteristic of their fully mycoheterotrophic nutrition.Seedlings and adults predominantly associated with saprotrophic fungi belonging to the Tulasnellaceae. While 13C and 2H stable isotope data revealed partial mycoheterotrophy of seedlings, it is unclear to what extent the fungal carbon supply is reduced in adult N. malipoensis. However, the 15N enrichment of mature N. malipoensis suggests partially mycoheterotrophic nutrition.Our data indicated a transition in mycorrhizal partners during ontogenetic development with decreasing dependency of N. malipoensis on fungal nitrogen and carbon. CONCLUSIONS: The divergence in mycorrhizal partners between N. malipoensis and A. nipponica indicates different resource acquisition strategies and allows for various habitat options in the earliest diverging orchid lineage Apostasioideae. While A. nipponica relies on the heterotrophic C gain from its ectomycorrhizal fungal partner and thus on forest habitats, N. malipoensis rather relies on own photosynthetic C gain as adult allowing it to establish in habitats as widely distributed as those where Rhizoctonia fungi occur.

Novel insights into orchid mycorrhiza functioning from stable isotope signatures of fungal pelotons.

Stable isotope signatures of fungal sporocarps have been instrumental in identifying carbon gains of chlorophyllous orchids from a fungal source. Yet, not all mycorrhizal fungi produce macroscopic sporocarps and frequently fungi of different taxa occur in parallel in orchid roots. To overcome this obstacle, we investigated stable isotope signatures of fungal pelotons extracted from orchid roots and compared these data to the respective orchid and reference plant tissues. Anoectochilus sandvicensis and Epipactis palustris represented specialized or unspecialized rhizoctonia-associated orchids. Epipactis atrorubens and Epipactis leptochila are orchids considered ectomycorrhiza-associated with different preferences for Basidio- and Ascomycota. 13 C enrichment of rhizoctonia pelotons was minor compared with plant tissues and significantly lower than enrichments of pelotons from ectomycorrhizal Epipactis species. 15 N values of pelotons from E. leptochila and E. atrorubens showed similar patterns as known for respective sporocarps of ectomycorrhizal Ascomycota and Basidiomycota, however, with an offset towards lower 15 N enrichments and nitrogen concentrations. Our results suggest an explicit fungal nutrition source of orchids associated with ectomycorrhizal fungi, whereas the low 13 C enrichment in rhizoctonia-associated orchids and fungal pelotons hamper the detection of carbon gains from fungal partners. 15 N isotopic pattern of orchids further suggests a selective transfer of 15 N-enriched protein-nitrogen into orchids.

Mycoheterotrophy in the wood-wide web.

The prevalence and potential functions of common mycorrhizal networks, or the 'wood-wide web', resulting from the simultaneous interaction of mycorrhizal fungi and roots of different neighbouring plants have been increasingly capturing the interest of science and society, sometimes leading to hyperbole and misinterpretation. Several recent reviews conclude that popular claims regarding the widespread nature of these networks in forests and their role in the transfer of resources and information between plants lack evidence. Here we argue that mycoheterotrophic plants associated with ectomycorrhizal or arbuscular mycorrhizal fungi require resource transfer through common mycorrhizal networks and thus are natural evidence for the occurrence and function of these networks, offering a largely overlooked window into this methodologically challenging underground phenomenon. The wide evolutionary and geographic distribution of mycoheterotrophs and their interactions with a broad phylogenetic range of mycorrhizal fungi indicate that common mycorrhizal networks are prevalent, particularly in forests, and result in net carbon transfer among diverse plants through shared mycorrhizal fungi. On the basis of the available scientific evidence, we propose a continuum of carbon transfer options within common mycorrhizal networks, and we discuss how knowledge on the biology of mycoheterotrophic plants can be instrumental for the study of mycorrhizal-mediated transfers between plants.

Fungal association and root morphology shift stepwise during ontogenesis of orchid Cremastra appendiculata towards autotrophic nutrition.

The chlorophyllous, terrestrial orchid Cremastra appendiculata from East Asia is unique concerning its fungal mycorrhiza partners. The initially mycoheterotrophic protocorms exploit rather specialized non-rhizoctonia saprotrophic Psathyrellaceae. Adult individuals of this orchid species are either linked to Psathyrellaceae being partially mycoheterotrophic or form mycorrhiza with fungi of the ubiquitous saprotrophic rhizoctonia group. This study provides new insights on nutrition mode, subterranean morphology and fungal partners across different life stages of C. appendiculata. We compared different development stages of C. appendiculata to surrounding autotrophic reference plants based on multi-element natural abundance stable isotope analyses (δ13C, δ15N, δ2H, δ18O) and total N concentrations. Site- and sampling-time-independent enrichment factors of stable isotopes were used to reveal trophic strategies. We determined mycorrhizal fungi of C. appendiculata protocorm, seedling and adult samples using high-throughput DNA sequencing. We identified saprotrophic non-rhizoctonia Psathyrellaceae as dominant mycorrhizal fungi in protocorm and seedling rhizomes. In contrast, the roots of seedlings and mature C. appendiculata were mainly colonized with fungi belonging to the polyphyletic assembly of rhizoctonia (Ceratobasidium, Thanatephorus and Serendipitaceae). Mature C. appendiculata did not differ in isotopic signature from autotrophic reference plants suggesting a fully autotrophic nutrition mode. Characteristic of orchid specimens entirely relying on fungal nutrition, C. appendiculata protocorms were enriched in 15N, 13C and 2H compared to reference plants. Seedlings showed an intermediate isotopic signature, underpinning the differences in the fungal community depending on their subterranean morphology. In contrast to the suggestion that C. appendiculata is a partially mycoheterotrophic orchid species, we provide novel evidence that mature C. appendiculata with rhizoctonia mycobionts can be entirely autotrophic. Besides an environmentally driven variability among populations, we suggest high within-individual flexibility in nutrition and mycobionts of C. appendiculata, which is subject to the ontogenetic development stage.